Analytical method for the simultaneous determination of dextromethorphan (1) and dextrorphan (2) in urine, based on solid-phase extraction of drug from acidified hydrolyzed biological matrix, were developed. The analytes (1 and 2) and the internal standard (levallorphan, 3, IS) were detected by high-performance liquid chromatography-mass spectrometry (HPLC-MS/MS) in positive ionization mode using a heated nebulizer (HN) probe and monitoring their precursor-->product ion combinations of m/z 272-->215, 258-->201, and 284-->201 for 1, 2, and 3, respectively, in multiple reaction monitoring mode. The analytes and IS were chromatographed on a Keystone Prism reverse phase (50 mm x 2.0 mm) 5 microm column using a mobile phases consisting of a 35/65 or 27/73 mixtures of methanol/water containing 0.1% TFA adjusted to pH 3 with ammonium hydroxide pumped at 0.4 ml/min for 1 and 2, respectively. The limits of reliable quantification of 1 and 2 were 2 and 250 ng/ml, respectively, when 1 ml of urine was processed. The absence of matrix effect was demonstrated by analysis of neat standards and standards spiked into urine extracts originating from five different sources. The linear ranges of the assay were 2-200 and 250-20,000 ng/ml for 1 and 2, respectively. Assay selectivity was evaluated by monitoring the "cross-talk" effects from other metabolites into the MS/MS channels used for monitoring 1, 2, and 3. In addition, an interfering peak originating from an unknown metabolite of 1 into the quantification of dextromethorphan was detected, requiring an effective chromatographic separation of analytes from other metabolites of 1. The need for careful assessment of selectivity of the HPLC-MS/MS assay in the presence of metabolites, and the assessment of matrix effect, are emphasized.

Concerns in pre-analytical handling of urine samples are discussed using a new KDR kinase inhibitor, 3-[5-(4-methanesulfonyl-piperazin-1-ylmethyl)-1H-indol-2-yl]-1H-quinolin-2-one (compound A), as an example of a case where high light sensitivity and low analyte recovery (high affinity for container surface) were found. The absence of these problems in plasma samples may be a result of the plasma protein content. Low recovery of the analyte from urine can be remedied by either changing the container or by using additives, such as bovine serum albumin (BSA) or non-ionic surfactant Tween-20. In the case of compound A, changing containers (polypropylene versus glass vial) or addition of BSA did bring analyte recovery up to 80%. However, the addition of 0.2% Tween-20 into urine quality controls (QCs) gave more than 95% analyte recovery, indicating effective reduction of analyte loss to the surface of containers. The urine assay using mixed-mode SPE and LC–MS/MS was not affected significantly by introducing Tween-20 into the samples. The mean SPE extraction recovery was 68.4% and matrix suppression of ionization on MS was less than 8% at all analyte concentrations. The linear range of the calibration curve was 0.5–400ng/mL on PE Sciex API 3000 LC–MS/MS system. The assay intraday accuracy and precision were 92.1–104.8% and <4.2% (%CV), respectively. Urine QC samples, containing 0.2% Tween-20, gave excellent recovery after three cycles of freeze and thaw. Since analyte loss to its urine container surface is not unique to compound A (M. Schwartz, W. Kline, B. Matuszewski, Anal. Chim. Acta 352 (1997) 299–307; A.L. Fisher, E. DePuy, T. Shih, R. Stearns, Y. Lee, K. Gottesdiener, S. Flattery, M. De Smet, B. Keymeulen, D.G. Musson, J. Pharm. Biomed. Anal. 26 (2001) 739–752), we suggest an evaluation of the potential problem in the early stages of urine assay development to ensure reliable quantitation of analytes. The addition of Tween-20 can serve as a useful analytical tool to other analytes with similar situations.

A fast and sensitive HPLC–MS/MS method, utilizing atmospheric pressure chemical ionization, for the determination of fexofenadine in human plasma is described. A deuterated analog, d6-fexofenadine is used as the internal standard (IS). Plasma samples are prepared using 96-well solid phase extraction with plates containing Waters Oasis HLB sorbent. The analytes are chromatographed on a Restek Ultra IBD column (3.2mm×50 mm, 3 μm) using a mobile phase consisting of a mixture of 90% acetonitrile and 10% 10 mM ammonium acetate buffer and 0.1% formic acid. Quantitation of the analyte is based on the response from the multiple reaction monitoring of the precursor to product ion pairs for fexofenadine (m/z 502→466) and d6-fexofenadine (m/z 508→472). The assay has been validated over the concentration range of 1–200 ng/ml based on the analysis of 0.5 ml aliquots of plasma. Within-day assay accuracy was between 97 and 102% of nominal, while within-day precision was better than 3.5% CV at all points on the standard curve. Analyte extraction recovery was better than 70% over the range of the standard curve. The method was found to be suitable for the analysis of human plasma samples obtained 24 h following the administration of a single 60 mg dose of fexofenadine.

Methods for the determination of a novel, ascomycin-based macrolide immunosuppressant in human plasma and whole blood are described. Following protein precipitation, the analyte and an internal standard were extracted from each matrix using solid phase extraction on an end-capped cyano column. The analytes were chromatographed on a Zorbax SB-CN analytical column (5.0 μm, 150×4.6 mm) with a mobile phase consisting of 70∶30∶0.1 v/v/v acetonitrile/ammonium acetate (10 mM)/formic acid. A tandem mass spectrometer equipped with an APCI interface was used as the detector. Multiple reaction monitoring using the parent→product ion combinations of m/z 1009→217 and 979→187 was used to detect the analyte and internal standard, respectively. Seven point calibration curves over the concentration range of 0.25–20 ng mL−1 yielded a linear response when a 1/yweighted linear regression model was employed. Based on the replicate analyses (n=5) of spiked standards, the withinday assay precision for both assays was better than 7.5% C.V. at all points on the calibration curves. The within-day accuracy for both assays was within 5% of nominal at all standard concentrations. The between-run precision of each of the assay, as calculated from the results of the analysis of quality control samples, was better than 5%. C.V.A special collection procedure for whole blood, in which samples were stored in the tubes that were used in the initial step of the assay procedure, was developed to eliminate assay error resulting from adsorption of the analyte to the sample storage tube.

A method for the determination of a macrolide immunosuppressant, L-732,531, in plasma is described. Plasma samples are extracted using cyano solid phase extraction columns. The extract is analyzed by normal phase high performance liquid chromatography with a cyano column and a mobile phase of hexane/2-propanol/water (780:220:3). Detection is based on fluorescence at an excitation wavelength of 276 nm and an emission wavelength of 330 nm. The limit of quantification of the assay is 1 ng/mL. The assay was validated in the concentration range of 1 – 200 ng/mL when 1 mL aliquots of plasma are extracted.

A method for the determination of rofecoxib in human plasma is described. After the addition of an internal standard, buffered (pH 5) plasma samples are extracted with hexane-methylene chloride (50:50, v/v). The extracts are evaporated to dryness and reconstituted in mobile phase. Upon exposure to UV light, the analyte was found to undergo a stilbene-phenanthrene-like photocyclization reaction with the resulting formation of a highly fluorescent species. Thus, the plasma extracts were analyzed via HPLC with post-column photochemical derivatization and fluorescence detection. The assay has been validated in the concentration range of 0.5-100 ng/ml using 1-ml samples. The method has been successfully utilized to support human clinical pharmacokinetic studies.

The HPLC/tandem mass spectrometric (LC/MS/MS) behavior of indinavir, an HIV protease inhibitor, in human urine is presented as an example of a case where endogenous matrix components were found to interfere with the ionization of the target analyte. The MS/MS system used for these experiments was equipped with a turbo ion spray LC interface. Results from two sample preparation procedures (direct dilution of urine vs urine extraction) and two chromatographic systems (low vs. high capacity factor (k')) for the analytes were compared. Additionally, the precision of the analysis that was achieved while using a stable isotope labeled internal standard is contrasted with the results obtained using an analog of indinavir as internal standard. The results obtained indicated that during development and validation of LC/MS/MS based assays the potential effect of co-eluting 'unseen' endogenous species should be evaluated to ensure that sample preparation and chromatography is adequate to overcome the matrix effect problems.

A method for the determination of 3-amino-5-ethyl-6-methyl-pyridin-2-one in human plasma is described. Buffered (pH 8) plasma samples are extracted with 7.5% 2-propanol in chloroform after the addition of an internal standard. The extracts are evaporated to dryness and reconstituted in mobile phase prior to analysis via HPLC with fluorescence detection (lambda(ex)=314 nm, lambda(em)=390 nm). The assay has been validated in the concentration range of 5-500 ng/ml when 1 ml samples are extracted. Application of the assay to the analysis of samples collected after oral dosing of a 2-pyridinone based specific HIV-1 reverse transcriptase inhibitor is demonstrated.